RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No.: 63/188,291 filed May 13, 2021, and entitled “TECHNIQUES FOR DNA RELEASING FROM MICROORGANISMS USING MICROFLUIDIC SYSTEMS," the entire contents of which is incorporated by reference herein.

BACKGROUND

[0002] Detection and identification of particles (e.g., bacterial and viral pathogens) present in cell containing solutions (e.g., blood, urine, CSF, mammalian cell culture, CHO cell matrix, CAR-T drug product, CAR-T specimen, CAR-NK drug product, body fluids, apheresis samples or samples related to immunotherapy), protein containing solutions (e.g., for pharmaceuticals during manufacturing, drug product, drug substance), analyte extraction from microbiome samples (including human microbiome samples), drug substance, drug product and drug samples along the manufacturing process, water, sterile fluids and other fluids is possible by employing isolation on cultural media and metabolic fingerprinting methods. Isozyme analysis, direct colony thin layer chromatography and gel electrophoresis techniques have been successfully applied for the detection of some bacterial pathogens. Immunoassay and nucleic acid-based assays are now widely accepted techniques, providing more sensitive and specific detection and quantification of bacteria.

[0003] Dielectrophoresis (DEP) relates to a force in an electric field gradient on objects having dielectric moments. DEP has shown promise for particle separation, but has not yet been applied in clinical settings, pharmaceutical quality assurance settings, or immunotherapy. DEP uses a natural or induced dipole to cause a net force on a particle in a region having an electric field gradient. The force depends on the Clausius-Mossotti factor associated with particle.

SUMMARY

[0004] Aspects of the technology described herein relate to techniques for releasing cellular content (e.g., DNA, RNA, proteins, membranes, organelles, etc.) from inside a cell captured on a surface of an electrode of a microfluidic device. [0005] In some embodiments, a method for releasing cellular contents from microorganisms in a fluid sample is provided. The method comprises activating at least one electrode disposed in a microfluidic passage of a microfluidic device to capture microorganisms in a fluid sample on a surface of the at least one electrode using dielectrophoresis, performing an action to release cellular contents from the captured microorganisms, and collecting the released cellular contents in a container coupled to an output of the microfluidic passage.

[0006] In one aspect, performing an action to release cellular contents from the captured microorganisms comprises causing the captured microorganisms to undergo lysis thereby releasing the cellular contents from the captured microorganisms. In one aspect, performing an action to release cellular contents from the captured microorganisms comprises passing a first fluid through the microfluidic passage, the first fluid including beads configured to cause mechanical damage to the captured microorganisms to release the cellular contents from the captured microorganisms.

[0007] In one aspect, the method further comprises controlling the at least one electrode to generate an electric field that causes the beads in the first fluid to be attracted to the at least one electrode via dielectrophoresis. In one aspect, passing the first fluid through the microfluidic passage comprises pumping the first fluid through the microfluidic passage. In one aspect, the method further comprises passing a second fluid through the microfluidic passage after passing the first fluid through the microfluidic passage, wherein the second fluid is a control solution that does not include the beads.

[0008] In one aspect, performing an action to release cellular contents from the captured microorganisms comprises passing a plurality of fluids including first and second fluids through the microfluidic passage, wherein passing the plurality of fluids through the microfluidic passage causes the captured microorganisms to undergo lysis thereby releasing the cellular contents from the captured microorganisms. In one aspect, passing the plurality of fluids through the microfluidic passage comprises pumping the plurality of fluids through the microfluidic passage. In one aspect, the method further comprises passing a third fluid through the microfluidic passage after passing the plurality of fluids through the microfluidic passage, wherein the third fluid is a control solution that does not cause lysis.

[0009] In one aspect, performing an action to release cellular contents from the captured microorganisms comprises passing a first fluid through the microfluidic passage, wherein passing the first fluid through the microfluidic passage causes the captured microorganisms to undergo chemical lysis thereby releasing the cellular contents from the captured microorganisms. In one aspect, passing the first fluid through the microfluidic passage comprises pumping the first fluid through the microfluidic passage. In one aspect, the method further comprises passing a second fluid through the microfluidic passage after passing the first fluid through the microfluidic passage, wherein the second fluid is a control solution that does not cause chemical lysis.

[0010] In one aspect, performing an action to release cellular contents from the captured microorganisms comprises heating a surface of the at least one electrode and/or the microfluidic channel to cause, at least in part, the captured microorganisms to undergo lysis to release cellular contents from the captured microorganisms. In one aspect, performing an action to release cellular contents from the captured microorganisms further comprises controlling the at least one electrode to generate an electric field that cause, at least in part, the captured microorganisms to undergo lysis to release cellular contents from the captured microorganisms. In one aspect, the method further comprises passing a fluid through the microfluidic passage through the microfluidic passage after performing the action to release cellular contents from the captured microorganisms, wherein the fluid is a control solution that does not cause the captured microorganisms to undergo lysis. In one aspect, the method further comprises measuring a temperature of the surface of the at least one electrode and/or inside the microfluidic channel, and heating the surface of the at least one electrode and/or the microfluidic channel based, at least in part, on the measured temperature of the surface of the at least one electrode and/or inside the microfluidic channel.

[0011] In one aspect, performing an action to release DNA from the captured microorganisms comprises applying an electric pulse to the at least one electrode to cause the captured microorganism undergo lysis thereby releasing cellular contents from the captured microorganisms. In one aspect, applying the electric pulse comprises applying a DC voltage to the at least one electrode. In one aspect, the method further comprises passing a fluid through the microfluidic passage through the microfluidic passage after performing the action to release cellular contents from the captured microorganisms, wherein the fluid is a control solution that does not cause the captured microorganisms to undergo lysis. In one aspect, the cellular contents include nucleic acids.

[0012] In some embodiments, a system for releasing cellular contents from microorganisms in a fluid sample is provided. The system comprises a microfluidic passage for receiving a fluid sample, the sample comprising microorganisms, at least one electrode disposed in the microfluidic passage, the at least one electrode configured to capture, when activated, the microorganisms onto a surface of the at least one electrode using dielectrophoresis, and a controller configured to perform an action to release cellular contents from the captured microorganisms.

[0013] In one aspect, the system is configured to cause the captured microorganisms to undergo lysis thereby releasing the cellular contents from the captured microorganisms. In one aspect, the system is configured to pass a first fluid through the microfluidic passage, the first fluid including beads configured to cause mechanical damage to the captured microorganisms to release the cellular contents from the captured microorganisms. In one aspect, the controller is further configured to control the at least one electrode to generate an electric field that causes the beads in the first fluid to be attracted to the at least one electrode via dielectrophoresis.

[0014] In one aspect, the system further comprises a pump configured to pass the first fluid through the microfluidic passage. In one aspect, the system is configured to pass a second fluid through the microfluidic passage after passing the first fluid through the microfluidic passage, wherein the second fluid is a control solution that does not include the beads. In one aspect, the system is configured to pass a plurality of fluids including first and second fluids through the microfluidic passage, wherein passing the plurality of fluids through the microfluidic passage causes the captured microorganisms to undergo lysis thereby releasing the cellular contents from the captured microorganisms. In one aspect, the system further comprises a pump configured to pass the plurality of fluids through the microfluidic passage. In one aspect, the system is configured to pass a third fluid through the microfluidic passage after passing the plurality of fluids through the microfluidic passage, wherein the third fluid is a control solution that does not cause lysis.

[0015] In one aspect, the system is configured to pass a first fluid through the microfluidic passage, wherein passing the first fluid through the microfluidic passage causes the captured microorganisms to undergo chemical lysis thereby releasing the cellular contents from the captured microorganisms. In one aspect, the system further comprises a pump configured to pass the first fluid through the microfluidic passage. In one aspect, the system is configured to pass a second fluid through the microfluidic passage after passing the first fluid through the microfluidic passage, wherein the second fluid is a control solution that does not cause chemical lysis.

[0016] In one aspect, the system is configured to heat a surface of the at least one electrode and/or the microfluidic channel to cause, at least in part, the captured microorganisms to undergo lysis to release cellular contents from the captured microorganisms. In one aspect, the controller is further configured to control the at least one electrode to generate an electric field that cause, at least in part, the captured microorganisms to undergo lysis to release cellular contents from the captured microorganisms. In one aspect, the system is configured to pass a fluid through the microfluidic passage through the microfluidic passage after performing the action to release cellular contents from the captured microorganisms, wherein the fluid is a control solution that does not cause the captured microorganisms to undergo lysis. In one aspect, the system is configured to measure a temperature of the surface of the at least one electrode and/or inside the microfluidic channel, and heat the surface of the at least one electrode and/or the microfluidic channel based, at least in part, on the measured temperature of the surface of the at least one electrode and/or inside the microfluidic channel.

[0017] In one aspect, the system is configured to apply an electric pulse to the at least one electrode to cause the captured microorganism undergo lysis thereby releasing cellular contents from the captured microorganisms. In one aspect, applying the electric pulse comprises applying a DC voltage to the at least one electrode. In one aspect, the system is configured to pass a fluid through the microfluidic passage through the microfluidic passage after performing the action to release cellular contents from the captured microorganisms, wherein the fluid is a control solution that does not cause the captured microorganisms to undergo lysis. In one aspect, the cellular contents include nucleic acids.

[0018] Some further aspects relate to a method for releasing DNA from a microorganism. The method comprises applying, with an electrode, at least one dielectrophoretic force which traps the microorganism on a surface of the electrode, injecting a solution comprising a plurality of beads into a microfluidic channel which houses the electrode, wherein injecting the solution causes the plurality of beads to collide with the microorganism, and collecting DNA released from the organism due to the colliding of the plurality of beads with the microorganism.

[0019] Some further aspects relate to a method for releasing DNA from a microorganism. The method comprises applying, with an electrode, at least one dielectrophoretic force which traps the microorganism on a surface of the electrode, injecting a solution into a microfluidic channel which houses the electrode, wherein injecting the solution causes the microorganism to undergo lysis, and collecting DNA released from the organism due to the lysis of the microorganism.

[0020] Some further aspects relate to a method for releasing DNA from a microorganism. The method comprises applying, with an electrode, at least one dielectrophoretic force which traps the microorganism on a surface of the electrode, adjusting a temperature of the surface of the electrode and/or a microfluidic channel which houses the electrode, and collecting DNA released from the organism due to adjusting the temperature. [0021] Some further aspects relate to a method for releasing DNA from a microorganism. The method comprising applying, with an electrode, at least one dielectrophoretic force which traps the microorganism on a surface of the electrode, applying an electric pulse to the microorganism while the microorganism is captured on the surface of the electrode, the electric pulse causing the microorganism to undergo lysis, collecting DNA released from the organism due to the lysis of the microorganism, and determining that a second portion of the sample is to be further processed.

BRIEF DESCRIPTION OF DRAWINGS

[0022] Various non-limiting embodiments of the technology will be described with reference to the following figures. It should be appreciated that the figures are not necessarily drawn to scale.

[0023] FIG. 1 schematically illustrates a system for detection and/or quantification of particles (e.g., bacteria or other microorganisms) in a sample, according to some embodiments; [0024] FIG. 2 illustrates a microfluidic system for detection and/or quantification of particles (e.g., bacteria or other microorganisms) in a sample in a sample, according to some embodiments;

[0025] FIG. 3 illustrates a static system for detection and/or quantification of particles (e.g., bacteria or other microorganisms) in a sample, according to some embodiments;

[0026] FIGS. 4A and 4B illustrate a schematic diagram of a first example method for releasing cellular contents from a microorganism in a sample by flushing a microfluidic device with a fluid containing beads, according to some embodiments;

[0027] FIGS. 5A and 5B illustrate a schematic diagram of a second example method for releasing cellular contents from a microorganism in a sample by applying a DNA extraction kit of one or more fluids to the microfluidic device, according to some embodiments;

[0028] FIG. 6A shows an electrode in a negative control prior to processing a sample, according to some embodiments; and

[0029] FIG. 6B shows an electrode with captured my coplasmas, according to some embodiments.

[0030] FIGS. 7 A and 7B illustrate a schematic diagram of a third example method for releasing cellular contents from a microorganism in a sample by applying, to the microfluidic device, fluids that cause chemical cell lysis of the microorganisms, according to some embodiments; [0031] FIGS. 8A and 8B illustrate a schematic diagram of a fourth example method for releasing cellular contents from a microorganism in a sample by heating the electrode of the microfluidic device to lyse the microorganisms, according to some embodiments;

[0032] FIGS. 9 A and 9B illustrate a schematic diagram of a fifth example method for releasing cellular contents from a microorganism in a sample by using an electrode of the microfluidic device to apply an electric pulse to lyse the microorganisms, according to some embodiments; and

[0033] FIG. 10 shows example results of an experiment conducted according to the fifth example method for releasing cellular contents from a microorganism shown in FIGS. 9A and 9B, according to some embodiments.

DETAILED DESCRIPTION

[0034] Aspects of the technology relate to various devices and techniques for releasing cellular contents from microorganisms using a microfluidic device. A microfluidic device may include one or more microfluidic channels through which a fluid can be passed for analysis. The microfluidic device may additional include one or more electrodes configured to generate an electric field to exert a di electrophoretic force on the microorganisms and/or other particles in the microfluidic channel. A microfluidic system may include such a microfluidic device and additional components for performing the analysis including, for example and without limitation, an input and/or output pump configured to move fluid through the microfluidic channel; source and/or sink containers for storing sample fluid, effluent, and/or waste; temperature control components such as a heating element, cooling element, and/or thermometer; detection components such as a sensor, camera, and/or mass spectrometer, etc.; one or more electrical signal generators for providing electrical signals to the electrodes; and/or a computer system to receive user input and operate the various components.

[0035] The techniques may include flushing a microfluidic device with a fluid containing beads to cause the beads to collide with microorganisms captured by the microfluidic device to cause mechanical damage to the microorganisms to release the cellular contents (e.g., DNA, RNA, proteins, membranes, organelles, etc.). The techniques may include pumping a series of solutions through a microfluidic device, where the series of solutions act to extract the cellular contents. The techniques may include flushing the microfluidic device with fluids that cause chemical cell lysis. The techniques may include adjusting a temperature of an electrode and/or a microfluidic channel of the microfluidic device to cause cell lysis. The techniques may include applying an electric pulse (e.g., in the range of several volts to tens of volts) to one or more electrodes to generate an electrical field that may cause microorganisms captured within the microfluidic device to undergo lysis to release cellular contents. These and other techniques described herein may be used in individually and/or in various combinations as described below and illustrated in the accompanying drawings.

[0036] FIG. 1 illustrates an example system 100 for detecting bacteria in a sample, in accordance with some embodiments. As shown in FIG. 1, the system 100 comprises a microfluidic device 104 in communication with a computing device 110.

[0037] The microfluidic device 104 may be any suitable device, examples of which are provided herein. In some embodiments, microfluidic device 104 is implemented as a microfluidic chip having one or more passages (e.g., microfluidic channels or chambers) through which a fluid sample 102 is provided for analysis. Although the term “microfluidic channel” or simply “channel” is used herein to describe a passage through which fluid flows through microfluidic device 104, it should be appreciated that a fluid passage having any suitable dimensions may be used as said channel, and embodiments are not limited in this respect. Microfluidic device 104 may comprise a single channel or multiple channels configured to receive a single sample 102 (e.g., to perform different analyses on the sample) or multiple channels configured to receive different samples for analysis. In embodiments having multiple channels, the microfluidic device may be configured to process the single sample or multiple samples in parallel (e.g., at the same or substantially the same time).

[0038] As described herein, sample 102 may include any fluid containing bacteria or other microorganism of interest. In some embodiments, the sample comprises a biological fluid such as saliva, urine, blood, water, any other fluid such as an environmental sample or potentially contaminated fluid, protein matrices, mammalian cell culture, immunotherapy drug product, cell and gene therapy drug product, cell and gene therapy drug sample, bacterial culture, growth media, active pharmaceutical ingredients, enzyme products, or substances used in biomanufacturing, etc.

[0039] As shown, microfluidic device 104 includes at least one electrode 106. The at least one electrode 106 may be configured to receive one or more voltages to generate positive and/or negative dielectrophoresis (DEP) force(s) that act on a sample arranged proximate to the at least one electrode. In some embodiments, the at least one electrode 106 may be configured to receive one or more voltages (e.g., one or more AC voltages) to generate at least one dielectrophoresis force that acts on the sample. The at least one DEP force may cause certain components of the sample to move relative to (e.g., be attracted to or repulsed from) a surface of the at least one electrode 106. For example, in the absence of an electric field, bacteria and other components of the sample 102 may move freely relative to the surface of the electrode. In the presence of the electric field, at least some components (e.g., bacteria) in the sample may be attracted to the electrode surface.

[0040] The small size of bacteria presents an obstacle to optical observation and quantification of bacteria in the sample. The inventors have recognized that activation of the at least one electrode 106 results in an electric field that may be used to selectively trap bacteria on the surface of the electrode(s). When used with an optical system, capturing bacteria on the surface of the electrode(s) may prevent the bacteria from moving in and out of focus of the optical system to enable real-time bacteria detection and quantification, a process referred to herein as “on-chip quantification.”

[0041] The electric field used to capture the bacteria concentrates the bacteria, which enables imaging with fluorescence microcopy or another optical detection technique. Accordingly, bacterial capture using the techniques described herein allows for detection and quantification of bacteria at significantly lower limits compared to some conventional methods. The ability to detect and/or quantify bacteria in a sample, even in small amounts, may be useful in applications including, but not limited to, biomanufacturing, gene therapy, analysis of patient samples, vaccine development and/or biothreat detection, and antibiotic susceptibility testing. [0042] For example, the at least one DEP force acting on the sample may cause bacteria (or certain bacteria) to separate from other components of the sample (e.g., via positive DEP). Bacteria in the sample may be attracted to the surface of the at least one electrode 106 allowing for enhanced detection and/or quantification, despite the small size and/or small amount of the bacteria in the sample. Although, microfluidic device 104 is illustrated as having a single electrode, it should be understood that in some embodiments, microfluidic device 104 comprises multiple electrodes arranged in any suitable configuration.

[0043] System 100 may further comprise a computing device 110 configured to control one or more aspects of microfluidic device 104. For example, computing device 110 may be configured to direct the sample 102 into a channel of the microfluidic device. In some embodiments, computing device 110 is configured to control the at least one electrode 106 to generate the at least one DEP force acting on the sample 102. In some embodiments, computing device 110 may cause one or more components of an optical system (not shown) to perform one or more of detection or quantification of the bacteria or other microorganisms in the sample. Non-limiting examples of a computing device 110 that may be used in accordance with some embodiments are further described herein. [0044] FIG. 2 illustrates an example microfluidic system 200 for detecting the presence of microorganisms (e.g., bacteria) in a sample, in accordance with some embodiments. System 200 includes microfluidic device 208 (e.g., indicated in FIG. 2 as a microfluidic chip) that includes one or more electrodes for generating DEP forces that act on a sample 204 provided as input to the system. Sample 204 may contain microorganisms for which separation, detection, enrichment, and/or quantification may be performed. The one or more electrodes may be arranged in any suitable configuration within the microfluidic device 208. For instance, in embodiments that include multiple electrodes, the electrodes may be arranged in one-dimension along the flow direction of the fluid, perpendicular to fluid flow direction or on a diagonal relative to the fluid flow direction. In some embodiments, a multidimensional (e.g., 2- dimensional, 3 -dimensional) array of electrodes may be used. For instance, a dense array of electrodes arranged both along the direction of fluid flow and perpendicular to the direction of fluid flow may be used.

[0045] As shown in FIG. 2, a flow system 202 is provided. The flow system 202 may provide a solution for transporting the sample 204 to the microfluidic device 208. A first pump 206 may be used to pump the solution and the sample 204 to the microfluidic device 208 at a predetermined flow rate. First pump 206 may be of any suitable type. In some embodiments, first pump 206 is omitted and sample 204 is manually loaded (e.g., using a pipette or capillary flow) as input to one or more channels of microfluidic device 208.

[0046] Microfluidic device 208 is configured to receive sample 204 for processing. Microfluidic device 208 may include one or more passages through which the sample 204 flows. The one or more passages may include at least one electrode formed therein or adjacent thereto. For instance, the at least one electrode may be formed within a passage. The at least one electrode, when activated, is configured to generate an electric field that acts on the sample 204 as it flows through the one or more passages. An electrical system 212 (e.g., a signal generator or controller) is configured to provide one or more voltages to the at least one electrode of the microfluidic device 208 to tune the properties of the electric field for capture of a particular microorganism or microorganisms of interest. Further aspects of the electrical system 212, including example protocols for operating the microfluidic device 208 are provided herein. [0047] Microfluidic system 200 may include an optical system 210 to facilitate analysis of the sample 204 by performing on-chip quantification. For example, optical system 210 may comprise one or more optical sensors (e.g., a red-green-blue camera) for viewing and/or imaging the sample. The optical sensor(s) may provide for enhanced detection and/or quantification of the captured microorganisms and/or the other components of the sample 204 relative to detection and quantification techniques that require separate culturing of captured microorganisms or an effluent sample from the device. Any suitable optical sensor(s) may be used. In some embodiments, the optical sensor(s) comprises a digital camera. In some embodiments, the optical sensor(s) includes a monochrome camera having a plurality of color filters. The monochrome camera may be configured to capture a plurality of monochrome images with different color filters, and a color image may be formed based on superposition of the plurality of monochrome images. For instance, the different color filters may include a red filter and a green filter, and the color image may be a superposition of a monochrome image captured with the red filter and a monochrome image captured with the green filter.

[0048] In some embodiments, the optical sensor(s) comprises electronic sensors including CMOS compatible technology. In some embodiments, the optical sensor(s) comprise fiber optics. However, any suitable optical sensor(s) may be used. In some embodiments, bacteria in the sample are stained (e.g., with a fluorescent dye) and the optical system 210 is configured to perform microscopy (e.g., fluorescence microscopy) of captured stained bacteria. In some embodiments, optical system 210 is configured to capture one or more images (e.g., color images) of the at least one electrode while the sample is flowing through the microfluidic device 208. In some embodiments, the detector comprises nanowire and/or nanoribbon sensors. In some embodiments, the field of view of the optical system 210 at a particular magnification is insufficient to capture the entire surface of the one or more electrodes. In such embodiments, the optical system 210 may be configured to capture multiple partially overlapping images that collectively cover the entire surface of the one or more electrodes. The multiple captured images may then be analyzed to detect and/or quantify the microorganisms of interest in the sample. [0049] Microfluidic system 200 also includes computer 230 configured to control an operation of optical system 210 and/or to receive images from optical system 210 and to perform processing on the received images (e.g., to count a number of microorganisms trapped by the microfluidic device 208). In some embodiments, the received images are analyzed to determine the number of microorganisms captured by the at least one electrode. For instance, microorganisms may be identified in the received images as spots (e.g., fluorescent spots) located on the edges of the electrodes. In this way, a captured target microorganism species may be differentiated from other components in the sample that are not captured and may appear as floating above the at least one electrode or located between electrodes.

[0050] After the sample 204 is processed by the microfluidic device 208 and/or optical system 210 to capture and/or quantify microorganism immobilized on the electrode(s), the sample 204 may be removed from the microfluidic device 208. For example, a second pump 216 may be provided for pumping the sample 204 out of the microfluidic device 208. The second pump 216 may be of any suitable type. In some embodiments, microfluidic system 200 comprises a flow sensor 214 for measuring a flow rate at which the sample 204 is removed from the microfluidic device 208. The flow sensor 214 and the second pump 216 may be in communication to control a flow rate at which the sample 204 is removed from the microfluidic device 208.

[0051] As described herein, microfluidic system 200 may be used for separating bacteria from other components (or for separating certain bacteria from other bacteria) in sample 204. Microfluidic system 200 comprises a waste region 218 arranged to receive other components of the sample 204 which have been separated from the bacteria by the microfluidic device 208 and subsequently removed from the sample 204, for example, using the second pump 216. In the description below, analysis of the fluid collected in waste region 218 may be referred to as analysis of the “effluent sample.” Microfluidic system 200 may further include effluent region 220 for receiving a purified version of sample 204 containing substantially only target microorganisms that were captured using microfluidic device 208.

[0052] In some embodiments, an amount of time needed to process a sample using microfluidic system 200 is substantially less than an amount of time required to process a sample using a conventional plate counting method (PCM) sample processing system. As shown in FIG. 2, processing a sample using system 200 may include at least three steps. In step 250, a sample is provided as input to microfluidic system 208 and bacteria are captured from the sample in the presence of an applied electric field. In step 260, automated on-chip quantification is performed, for example, using computer 230 to analyze one or more images recorded by optical system 210. In step 270, further analysis may be performed on waste 218 and/or effluent sample 220, as desired. In sum, the entire process for detecting and/or quantifying microorganisms in a sample using system 200 may take on the order of minutes or an hour to a few hours, which is substantially faster than the multiple days (e.g., 1 to 14 days) typically required to process samples using PCM.

[0053] In some embodiments, rather than pumping sample 204 through one or more passages through which the sample flows, sample 204 may be manually provided as input to microfluidic device 208 for analysis. For instance, one or more droplets of sample 204 may be provided as input to microfluidic device 208 using a pipette or other suitable technique. In such embodiments, the sample is analyzed in a “static” condition rather than in a condition in which microorganisms are captured by the at least one electrode as the sample flows past the electrode(s) (e.g., as in the case of microfluidic system 200 as shown in FIG. 2). FIG. 3 illustrates a microfluidic system 300 for detecting microorganisms in a sample, according to some embodiments. As shown, microfluidic system 300 may include many of the same components as microfluidic system 200, but may omit certain components of the microfluidic system 200, such as the first pump 206, which are not needed when the sample is manually provided as input to the microfluidic device 308 (indicated in FIG. 3 as a static microfluidic chip).

[0054] Detection and/or measurement of certain cellular contents such as DNA may be useful for certain types of analysis such as detecting mycoplasma and/or microbial contamination. Mycoplasma is a genus of bacteria that can infect various cells in vivo and/or in vitro. Mycoplasma contamination may afflict cell cultures used in pharmaceutical research and manufacturing for producing large molecules and/or cell therapies. Existing techniques for testing for mycoplasma contamination have significant drawbacks. Culture-based methods may take days or weeks to produce results. Molecular techniques may require complex sample preparation to extract DNA. For example, molecular techniques may rely on separating mammalian cells from a sample, and then extracting DNA to detect mycoplasma. Detecting a low level of mycoplasma DNA may indicate a low concentration of mycoplasma in a complex sample matrix, with a high concentration of DNA coming from other species and organisms. [0055] Microbial contamination may affect sterility testing and/or virus testing. Such testing may require detecting a small concentration of an organism that causes contamination in a complex sample matrix that may contain DNA from one or more other species.

[0056] Disclosed herein are various devices and techniques for releasing cellular contents from microorganisms using a microfluidic device. Such devices and techniques may be particularly beneficial for analysing a sample in which a concentration of the DNA to be detected is relatively low. The devices and techniques may apply to detection and/or analysis of various kinds of cellular contents (e.g., DNA, RNA, proteins, membranes, organelles, etc.) from various types of cells (e.g., animal, plant, bacteria, yeast, mold, viruses, etc.).

[0057] Microorganisms may be captured by an electrode of a microfluidic device as described herein and/or as described in U.S. Patent Application No. 17/139,384, entitled “HIGH-EFFICIENCY BACTERIA CAPTURE AND QUANTIFICATION SYSTEM AND METHODS” filed on December 31, 2020, and subsequently issued as U.S. Patent No. 11,198,843 on December 14, 2021, which is hereby incorporated by reference in its entirety. [0058] Several techniques for releasing cellular contents from microorganisms captured by an electrode of a microfluidic device are provided herein. A first example technique includes flushing the microfluidic device with a fluid containing beads as described with reference to FIGS. 4A and 4B. A second example technique includes applying a DNA extraction kit of one or more fluids to the microfluidic device as described with reference to FIGS. 5 A and 5B. A third example technique includes applying fluids that cause chemical cell lysis of the microorganisms as described with reference to FIGS. 7A and 7B. A fourth example technique includes generating an electric field and/or heat using an electrode of the microfluidic device to lyse microorganisms as described with reference to FIGS. 8A and 8B. A fifth example technique includes using an electrode of the microfluidic device to apply an electric pulse to the microorganisms as described with reference to FIGS. 9A and 9B. These and other techniques are not mutually exclusive and may be used in individually and/or in various combinations with each other or with other techniques described elsewhere.

[0059] FIGS. 4A and 4B illustrate a schematic diagram of a first example method for releasing cellular contents from a microorganism in a sample by flushing a microfluidic device 104 with a fluid containing beads, according to some embodiments. When flushed through the microfluidic device, beads 403 may collide with microorganisms 401 captured on the surface of the electrode 409 of the microfluidic device 104, damaging the microorganisms 401 and causing cell lysis. To enhance this effect, the electrode 409 may generate an electric field that exerts a dielectrophoretic force on the beads 403 and/or microorganisms 401. The dielectrophoretic force may attract the beads 403 to the electrode 409 and cause collisions with the microorganisms 401. The mechanical damage to the microorganisms 401 may release cellular contents 402 such as DNA, RNA, proteins, organelles, etc. The beads 403 and cellular contents 402 may be flushed to an outlet of the microfluidic device for collection in an effluent sample container 414. [0060] FIG. 4A shows a first phase 400 of the method as viewed through a cross section 406 of the microfluidic device 104. In the first phase 400, microorganisms 401 are captured using the techniques described previously with reference to FIGS. 1 through 3. A fluid sample containing bacteria or other microorganisms 401 of interest may be contained in an influent container 404 and delivered to an inlet 405 of the microfluidic device 104 (e.g., via a pump of the microfluidic system 200). In some implementations, the fluid may be drawn through the microfluidic device 104 by a pump connected to an outlet 413 of the microfluidic device 104. The sample fluid may travel through the microfluidic device 104 in a direction 410. The electrode 409 may be activated by applying a time-varying (AC) electrical signal (e.g., a voltage) to the electrode. The electrical signal applied to the electrode 409 may generate an electric field that exerts a dielectrophoretic force on the microorganisms 401. The dielectrophoretic force may attract the microorganisms 401 to the electrode 409, pulling them along a trajectory 408. Captured microorganisms 411 remain adhered on or near the electrode 409, despite continued fluid flow through the microfluidic device 104.

[0061] FIG. 4B shows a second phase 450 of the method. In the second phase 450, one or more actions may be performed to release cellular contents from the captured microorganisms 411. In some implementations, a solution containing beads 403 may be pumped through the microfluidic device 104 (e.g., in the direction 410). The beads 403 may collide with the captured microorganisms 411. The collisions may cause mechanical damage to the captured microorganisms 411 resulting in cell lysis and release of the cellular contents 402. The released cellular contents 412, as well as the beads 403 and other fluid in the microfluidic channel may flow into an effluent sample container 414 from an outlet 413 of the microfluidic device. In some implementations, a control solution that does not include beads may be passed through the microfluidic device 104 to facilitate collection of the cellular contents 402 in effluent sample container 414. In some implementations, the effluent may be analyzed further using the microfluidic device. In some implementations, the collisions between the beads 403 and the microorganisms 401 may be enhanced by attracting the beads 403 to the electrode 409 using a dielectrophoretic force. Beads 407 moving towards the electrode 409 may have more frequent and/or more energetic collisions with the captured microorganisms 411 than the beads 403 traveling in the direction 410 of fluid flow.

[0062] FIGS. 5A and 5B illustrate a schematic diagram of a second example method for releasing cellular contents from a microorganism in a sample using a DNA extraction kit that includes one or more fluids provided as input to the microfluidic device 104, according to some embodiments. A first phase of the method may be similar to the first phase 400 of the first example method. A fluid sample containing bacteria or other microorganisms 401 of interest may be contained in an influent container 404 and delivered to an inlet 405 of the microfluidic device 104 (e.g., via a pump of the microfluidic system 200). The electrode 409 may be controlled to generate an electric field that exerts a dielectrophoretic force on the microorganisms 401, capturing them on or near the electrode 409.

[0063] FIG. 5A shows a second phase 500 of the method. A series of solutions 503 (e.g., from one or more influent containers 504) may be pumped through the microfluidic device 104. The series of solutions 503 may include, for example, solutions from a DNA extraction kit. The solutions 503 may be configured to extract cellular contents 402 from the captured microorganisms 411.

[0064] In the third phase 550, shown in FIG. 5B, the extracted cellular contents 512 may exit the microfluidic device 104 via the outlet 413, from which they may be collected in an effluent sample container 514. In some implementations, the microfluidic device 104 may be flushed with a control solution — e.g., one that does not cause cellular lysis — to facilitate collection of the extracted cellular contents 512 in the effluent sample container 514. In some implementations, the effluent may be analyzed further using the microfluidic device and/or other techniques/instruments .

Example 1: Capture of A. laidlawii from mammalian cell culture media using a microfluidic system

[0065] In this example, an experiment performed according to the second method described above. Mycoplasma contamination of biopharmaceutical products and of cell-based medicinal products poses a potential health threat to patients. My coplasmas can affect cell cultures without visible effects with standard light microscopy, which is an undesirable factor in the pharmaceutical and cell therapy industry. Therefore, manufacturers may be obligated (e.g., by regulatory agencies) to test their biopharmaceutical products to confirm the absence of my coplasmas in released products. For example, such agencies may have guidelines that regulate validation of testing methods such as rapid NAT (nucleic acid amplification techniques). Lack of harmonization of rapid mycoplasma test regulations pose a challenge for biomanufacturing.

[0066] Mycoplasmas include members of the bacterial class Mollicutes. Mollicutes are characterized by the lack of a cell wall and a small genome size (0.5 -2.2 mbp), and their life is host-dependent. Mycoplasmas can grow and propagate under aerobic or anaerobic conditions. Depending on the species, in liquid media mycoplasmas can grow as single cells (e.g., M. arthritidis ) or form in aggregates (e.g., A. laidlawii, M. fermentans). The lack of a cell wall makes mycoplasmas resistant to cell wall-targeting antibiotics such as penicillin.

[0067] Currently, to detect mycoplasmas, compendial testing methods like Culture Method and the Indicator Cell Culture Method; and non-compendial testing methods like Direct DNA staining or Enzyme -based methods are used. A separate group of testing methods include NAT- based methods based on nucleic acids detection performed by PCR. These methods include, but are not limited to, such rapid tests like My coTOOL (Roche), MycoSEQ (ThermoFisher Sci), Venor ^™ GeM Mycoplasma Detection Kit and LookOut ^® Mycoplasma PCR Detection Kit.

[0068] Using a microfluidic device, such as the microfluidic device 104 described herein, mycoplasmas may be captured directly from mammalian cells, which may reduce the time to detect contamination from hours or days to minutes. PBS (buffer solution) [0069] Control sample media was prepared in a sterile phosphate buffered saline (PBS) pH 7.4 without calcium chloride and magnesium chloride (Life Technologies, USA) diluted 1:1000 UltraPure Distilled Water (DI water, Life Technologies, USA) in aseptic condition. The conductivity of the PBS 1:1000 in DI water was in the range 19-23 µS/cm and was measured at RT using pH/mV/conductivity meter Accumet® XL200. Aliquots of diluted PBS were stored at 4°C. Mollicutes [0070] Acholeplasma laidlawii-23206-TTR™ Strain PG8 were stored in -80°C according to manufacture storage conditions. An original sample was thawed in ice, mixed well and aliquoted at 50 µL to cryotubes and frozen back to -80°C. There was no propagation or culture of A. laidlawii. To run the experiment, an exact number of tubes with 50µL of mycoplasma was thawed mixed and spiked to the tested samples or control buffer. All experimental work was performed in room temperature and Class II biosafety cabinet. CHO Conditioned Media [0071] The CHO culture was sampled from the bioreactor, aliquoted to 2- or 15-mL sterile tube and stored in 4°C. Before processing on the microfluidic system 200, CHO cultured medium was incubated for 30 min in RT, then diluted 1:100 in a sterile UltraPure Distilled Water to achieve a working range of conductivity. The conductivity of the diluted CHO cultured medium in UltraPure DI water was in the range 130-150 µS/cm and was measured at room temperature (RT) using pH/mV/conductivity meter ACCUMET XL200. Aliquots of diluted PBS were stored at 4°C. DNA extraction [0072] DNA extraction from influent (inlet sample) and effluent (outlet sample) was performed using AME PrepSEQ (ThermoFisher Sci, USA). qPCR [0073] The presence of A. laidlawii DNA in analyzed samples was confirmed using MycoSEQ closed PCR real-time system (ThermoFisher Sci, USA). A. laidlawii staining with fluorescent dye [0074] To optically visualize all bacterial strains, bacteria ware stained with a green, fluorescent dye SybrGreen I (ThermoFisher, USA) according to the manufacturer’s protocol. To 1 mL of suspended bacteria in UltraPure DI water, 1 µL of SybrGreen I was added. [0075] The sample was mixed by vortexing for 5 seconds and then incubated 30 min in room temperature (RT) in darkness. After incubation time, the sample was mixed once again and was ready to process.

Sample preparation

[0076] Briefly, directly before the experiment, A. laidlawii- 23206-TTR™ Strain PG8 was thawed from -80°C mixed, transferred to microcentrifuge tube, and spun down for 5 min at 5000 rpm in room temperature (RT). Supernatant was gently discarded. Pellet of A. laidlawii was resuspended in 1 mL of UltraPure DI water and 1 µL of SybrGreen was added. Sample was well mixed and incubated for at least 15 min in darkness. When incubation was done, 10µL of CHO processed media was added and well mixed. Finally, using stained stock of A. laidlawii, 10 mL of A. laidlawii diluted 1:1000 in CHO processed media 1:100 in UltraPure DI Water was prepared. Such a prepared sample was ready for processing using a microfluidic system (e.g., the microfluidic system 200) in a flow condition.

[0077] A control sample was prepared in the same manner, except that the A. laidlawii was suspended in PBS and not in the CHO processed media. In total, four samples were prepared to test on the microfluidic system and both influent and effluent of each sample were further analyzed by qPCR to confirm A. laidlawii DNA in spiked samples, as shown in Table 1.

Table 1: Description of samples

Microfluidic System.

[0078] All tests were performed at RT using a microfluidic system similar to that shown in FIG. 2. To evaluate visually the response of A. laidlawii to the electric field, the bacterial sample was stained with SybrGreen I during mycoplasma preparation. Samples containing suspended mycoplasma were processed through a flow chip.

Electronic conditions for Example 1

[0079] When sample began processing through the microfluidic system, an electric field having characteristics of 25 V peak to peak (Vpp) was applied at 2 MHz. Captured bacteria on the spiral electrodes of the microfluidic device were visualized using an optical system.

Capture A. laidlawii from CHO processed media

[0080] In this example, it was demonstrated that by using a microfluidic system and modulating the electric field (voltage, frequency) A. laidlawii can be captured from both a buffer solution and CHO processed media, and the bacteria can be released for further DNA analysis.

A. laidlawii was stained with SybrGreen I dye and suspended in FS standard buffer. Such prepared samples (spiked and un-spiked) were processed in flow conditions using the microfluidic system 200. When electrical conditions of 25 Vpp and frequency 2 MHz were reached, A. laidlawii, despite its small size and lack of cell wall, responded to the electric field in a characteristic manner. For example, the sample responded by forcing loosely floating bacteria (when electric field is off) from any area of the microfluidic device to the edges of electrodes (when electric field is on) in a characteristic positive dielectrophroesis (pDEP) response. When the electric field was off, A. laidlawii were then released from edges of electrodes, effluent was collected, DNA extracted, and qPCR performed to confirm presence of A. laidlawii only in spiked samples. The qPCR analysis for control samples, buffer solution and CHO processed media without spiked A. laidlawii, gave negative results, indicating no DNA was detected.

[0081] FIG. 6A shows an electrode in a negative control scan prior to processing a sample, according to some embodiments. The microfluidic device 104 was scanned (e.g., by the optical system 210) prior to the sample passing through the microfluidic device. The image 600 (magnified; e.g., ~40x) shows little or no signal indicating captured mycoplasmas.

[0082] FIG. 6B shows an electrode with captured mycoplasmas, according to some embodiments. The microfluidic device 104 was scanned after receiving CHO processed media with spiked and stained with SybrGreen I A. laidlawii. The image 650 shows visible green spots indicating mycoplasmas captured to the edges of the electrode spirals.

[0083] In this example, it was shown that by using a microfluidic system (e.g., the microfluidic system 200) A. laidlawii bacteria can be captured and quantified from CHO processed media. Additionally, qPCR analysis confirmed that A. laidlawii DNA were detected only in the spiked sample. No false negative results were observed for spiked samples and no false positive results were observed for un-spiked control samples. The obtained results showed that the use of the microfluidic system combined with subsequent qPCR can specifically detect A. laidlawii contamination in mammalian cell cultured media and significantly reduce time to the final results from hours to the days (up to 28 days) when compared to compendial methods (e.g., cultured methods).

[0084] FIGS. 7 A and 7B illustrate a schematic diagram of a third example method for releasing cellular contents from a microorganism in a sample by applying, to the microfluidic device 104, fluids that cause chemical cell lysis of the microorganisms, according to some embodiments. The method is similar to the second method described above. In the third method, however, the solutions 503 are replaced with a fluid 703 that causes chemical cell lysis. The cell lysis causes release of cellular contents 402.

[0085] A first phase of the method may be similar to the first phase 400 of the first example method. In the first phase, a sample containing bacteria or other microorganisms 401 of interest may be delivered, from an influent container 704, to the microfluidic device 104 via the inlet 405. The electrode 409 may be activated to produce an electric field that exerts a di electrophoretic force on the microorganisms 401, capturing them on or near the electrode 409. [0086] FIG. 7 A shows a second phase 700 of the method. The fluid 703 may be pumped (e.g., from an influent container 704) through the microfluidic device 104. The fluid 703 may cause chemical cell lysis of captured microorganisms 411.

[0087] In the third phase 750, shown in FIG. 7B, the released cellular contents 712 may exit the microfluidic device 104 via the outlet 413 and may be collected in an effluent sample container 714. In some implementations, the microfluidic device 104 may be flushed with a control solution — e.g., one that does not cause cellular lysis — to facilitate collection of the released cellular contents 712 in the effluent sample container 714.

[0088] FIGS. 8A and 8B illustrate a schematic diagram of a fourth example method for releasing cellular contents from a microorganism in a sample by heating the electrode of the microfluidic device 104 to lyse the microorganisms, according to some embodiments. A first phase of the method may be similar to the first phase 400 of the first example method. In the first phase, a fluid sample containing bacteria or other microorganisms 401 of interest may be contained in an influent container 404 and delivered to an inlet 405 of the microfluidic device 104 (e.g., via a pump of the microfluidic system 200). The electrode 409 may generate an electric field that exerts a dielectrophoretic force on the microorganisms 401, thereby capturing the microorganism 401 on or near the electrode 409.

[0089] FIG. 8A shows a second phase 800 of the method. A magnitude of the electrical signal provided to the electrodes 409 may be increased, as shown by the waveform 820. The higher-amplitude electrical signal may cause heating of the electrode 409 and/or an increase in the strength of the electrical field. In some implementations, a heating element of the microfluidic device 104 may be heated with a direct current and/or alternating current electric signal. The microfluidic device 104 may include a thermal sensor to measure a temperature of the electrode 409, microfluidic channel, and/or a fluid contained therein. The generated heat and/or electric field may cause lysing of captured microorganisms 411. Lysing of the captured microorganisms 411 may release their cellular contents 402.

[0090] In the third phase 850, shown in FIG. 8B, the released cellular contents 812 may exit the microfluidic device 104 via the outlet 413, from which they may be collected in an effluent sample container 814. In some implementations, the microfluidic device 104 may be flushed with a control solution to facilitate collection of the released cellular contents 812 in effluent sample container 814.

[0091] In some implementations, the method may include determining a temperature of the surface the electrode 409. The system 200 may determine the temperature either directly (e.g., with a probe or other instrument), or indirectly (e.g., by calibrating the electrode 409 and the electrical signal). In some implementations, the method may include implementing a feedback loop to maintain the temperature of the electrode surface (and/or the microfluidic channel) within a predetermined range.

[0092] FIGS. 9 A and 9B illustrate a schematic diagram of a fifth example method for releasing cellular contents from a microorganism in a sample by using an electrode of the microfluidic device 104 to apply an electric pulse to lyse the microorganisms, according to some embodiments. A first phase of the method may be similar to the first phase 400 of the first example method. In the first phase, a fluid sample containing bacteria or other microorganisms 401 of interest may be contained in an influent container 404 and delivered to an inlet 405 of the microfluidic device 104 (e.g., via a pump). The electrode 409 may generate an electric field that exerts a di electrophoretic force on the microorganisms 401, thereby capturing the microorganisms 401 on or near the electrode 409.

[0093] FIG. 9A shows a second phase 900 of the method. The system 200 may supply an electrical “kill pulse” to the electrodes 409, as shown by the waveform 920. For example, a 1MHz, 50V _PP signal may be used to capture the microorganisms 401 on or near the electrode 409. Subsequently, a DC electrical pulse of approximately 30V provided to the electrode 409 may cause the captured microorganisms 411 to undergo lysis, thereby releasing their cellular contents 402. [0094] In the third phase 950, shown in FIG. 9B, the released cellular contents 912 may exit the microfluidic device 104 and may be collected in an effluent sample container 914. In some implementations, the microfluidic device 104 may be flushed with a control solution to facilitate collection of the released cellular contents 912 in the effluent sample container 914.

[0095] The following is an example of an experiment performed according to the fifth method. Results of the experiment are shown in FIG. 10.

Example 2: DNA releasing from microorganisms using a microfluidic system and electric pulse [0096] Aspects of the technology described herein relate to lysing of microorganisms and releasing of cellular contents by applying a single electric killing pulse to the previously captured microorganisms. Using this two-step process which includes (i) capturing the target microorganisms and then (ii) lysing the microorganisms, thereby releasing cellular contents (e.g., nucleic acids) and collecting effluent containing the cellular contents, may significantly reduce the time from hours to minutes for extraction/precipitation of, for example, DNA, when compared to conventional methods of DNA extraction, especially for gram-positive microorganisms where detergent-enzymatic lysis may be helpful or necessary to increase DNA yield.

General Protocols

PBS

[0097] The bacterial strain used in DNA release experiments was suspended in sterile phosphate buffered saline (PBS) pH 7.4 without calcium chloride and magnesium chloride (Life Technologies, USA) diluted 1:1000 UltraPure Distilled Water (DI water, Life Technologies, USA). The conductivity of the PBS 1:1000 in DI water was in the range 19-23 μS/cm and was measured at room temperature (RT) using pH/mV/conductivity meter Accumet® XL200. Aliquots of diluted PBS were stored at 4°C.

Bacterial strain

[0098] E. faecalis were propagated and cultured on LB medium supplemented with Tetracycline because this strain carries a tetracycline resistance gene. All experimental work was performed in room temperature and Class II biosafety cabinet.

Sample preparation

[0099] The E. faecalis strain was obtained from ATCC and cultured on LB/tet medium agar plates at 37°C in aerobic conditions. A day before the experiment, the E. faecalis was re- streaked on LB/tet medium agar plates by the progressive dilutions of an inoculum (from a single colony) on agar plates using a sterile inoculation loop. From an overnight culture, a large scoop of bacteria (avoiding taking bacteria from a biofilm area on the plates) was taken using sterile inoculation loop and suspended in 10 mL of PBS 1:1000. A stock sample concentration of 10 ¹⁰ -10 ⁹ bacteria/mL was determined using an OD (optical density, C08000 Cell Density Meter, Biowave) meter at 600 nm. The stock sample was diluted by serial lOx dilution to the final bacterial concentration of influent (input sample which is processed on the microfluidic system 200) up to 10 ⁸ bacteria/mL. All experiments were conducted at room temperature. To prepare stock and experimental samples buffer and media were warmed up to room temperature prior to use.

Released DNA analysis

[0100] After releasing of DNA from lysed bacteria using an electric killing pulse, 1 mL of effluent (outlet sample containing nucleic acids suspension in PBS 1:1000 in UltraPure DI water, DNAse and RNase free) was collected and transferred to the ice. To concentrate released DNA, the sample was purified using standard phenol/chloroform extraction and ethanol precipitation. Finally, precipitated DNA was suspended in 50 µL of TE buffer (Tris/EDTA). Concentration of nucleic acids were determined using Qubit Fluorometers (ThermoFisher Sci, USA). Additionally, presence of DNA was confirmed using 2100 Expert High Sensitivity DNA assay (Agilent Technologies, Inc., USA).

[0101] The capture and lyse of E. faecalis was performed using a microfluidic system (e.g., a microfluidic system similar to that shown in FIG. 2) in a flow condition and was performed at room temperature. The sample was processed through a flow chip.

[0102] The electronic conditions for bacteria capture in Example 2 were as follows: (i) To capture bacteria, electric field 50 V peak to peak (Vpp) was applied at 1 MHz frequency, (ii) To lyse captured E. faecalis and release nucleic acids therefrom, a kill pulse of 30V DC was applied.

Release of nucleic acids from E. faecalis by lysis of bacteria using electric killing pulse [0103] In this study it was demonstrated that using a flow-based microfluidic device in combination with modulation of electric field characteristics (voltage, frequency) enabled first capture and then release of nucleic acids by lysis of tested bacteria using an electric killing pulse. E. faecalis was suspended in tested buffer (PBS 1 : 1000 in DI water) and processed through the microfluidic system in flow conditions. When certain voltage (50 Vpp) and frequency (1 MHz) conditions were reached, all bacteria were captured on the surface of an electrode of the microfluidic system. To lyse bacteria and release nucleic acids from captured microorganisms, a killing pulse of 30V DC was applied. Lysed bacteria (mixed with cell debris and nucleic acids in PBS) were collected. Phenol/chloroform extraction and ethanol precipitation were performed to reduce the volume of the sample and concentrate the collected DNA. Based on Qubit Fluorometer analysis, the 30VDC kill pulse was strong enough to lyse bacteria and release nucleic acids with 0.5 ng/mL. Presence of DNA was confirmed using 2100 Expert High Sensitivity DNA assay, as well (see electropherogram in FIG. 10).

[0104] FIG. 10 shows example results 1000 of the Example 2 experiment conducted according to the fifth example method for releasing cellular contents from a microorganism shown in FIGS. 9A and 9B, according to some embodiments. It was shown in this example that the electric killing pulse was able to rapidly lyse gram positive bacteria and release nucleic acids in enough quantity for further analysis, such as PCR, RT-PCR or sequencing, without any additional processing steps, thereby substantially simplifying existing techniques.

[0105] Having thus described several aspects and embodiments of the technology set forth in the disclosure, it is to be appreciated that various alterations, modifications, and improvements will readily occur to those skilled in the art.

[0106] Such alterations, modifications, and improvements are intended to be within the spirit and scope of the technology described herein. For example, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the embodiments described herein. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described. In addition, any combination of two or more features, systems, articles, materials, kits, and/or methods described herein, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present disclosure.

[0107] The above-described embodiments can be implemented in any of numerous ways. One or more aspects and embodiments of the present disclosure involving the performance of processes or methods may utilize program instructions executable by a device (e.g., a computer, a processor, or other device) to perform, or control performance of, the processes or methods. In this respect, various inventive concepts may be embodied as a computer readable storage medium (or multiple computer readable storage media) (e.g., a computer memory, one or more floppy discs, compact discs, optical discs, magnetic tapes, flash memories, circuit configurations in Field Programmable Gate Arrays or other semiconductor devices, or other tangible computer storage medium) encoded with one or more programs that, when executed on one or more computers or other processors, perform methods that implement one or more of the various embodiments described above. The computer readable medium or media can be transportable, such that the program or programs stored thereon can be loaded onto one or more different computers or other processors to implement various ones of the aspects described above. In some embodiments, computer readable media may be non-transitory media.

[0108] The terms “program” or “software” are used herein in a generic sense to refer to any type of computer code or set of computer-executable instructions that can be employed to program a computer or other processor to implement various aspects as described above. Additionally, it should be appreciated that according to one aspect, one or more computer programs that when executed perform methods of the present disclosure need not reside on a single computer or processor but may be distributed in a modular fashion among a number of different computers or processors to implement various aspects of the present disclosure.

[0109] Computer-executable instructions may be in many forms, such as program modules, executed by one or more computers or other devices. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Typically, the functionality of the program modules may be combined or distributed as desired in various embodiments.

[0110] Also, data structures may be stored in computer-readable media in any suitable form. For simplicity of illustration, data structures may be shown to have fields that are related through location in the data structure. Such relationships may likewise be achieved by assigning storage for the fields with locations in a computer-readable medium that convey relationship between the fields. However, any suitable mechanism may be used to establish a relationship between information in fields of a data structure, including through the use of pointers, tags or other mechanisms that establish relationship between data elements.

[0111] The above-described embodiments of the present technology can be implemented in any of numerous ways. For example, the embodiments may be implemented using hardware, software or a combination thereof. When implemented in software, the software code can be executed on any suitable processor or collection of processors, whether provided in a single computer or distributed among multiple computers. It should be appreciated that any component or collection of components that perform the functions described above can be generically considered as a controller that controls the above-described function. A controller can be implemented in numerous ways, such as with dedicated hardware, or with general purpose hardware (e.g., one or more processor) that is programmed using microcode or software to perform the functions recited above and may be implemented in a combination of ways when the controller corresponds to multiple components of a system.

[0112] Further, it should be appreciated that a computer may be embodied in any of a number of forms, such as a rack-mounted computer, a desktop computer, a laptop computer, or a tablet computer, as non-limiting examples. Additionally, a computer may be embedded in a device not generally regarded as a computer but with suitable processing capabilities, including a Personal Digital Assistant (PDA), a smartphone or any other suitable portable or fixed electronic device.

[0113] Also, a computer may have one or more input and output devices. These devices can be used, among other things, to present a user interface. Examples of output devices that can be used to provide a user interface include printers or display screens for visual presentation of output and speakers or other sound generating devices for audible presentation of output. Examples of input devices that can be used for a user interface include keyboards, and pointing devices, such as mice, touch pads, and digitizing tablets. As another example, a computer may receive input information through speech recognition or in other audible formats.

[0114] Such computers may be interconnected by one or more networks in any suitable form, including a local area network or a wide area network, such as an enterprise network, and intelligent network (IN) or the Internet. Such networks may be based on any suitable technology and may operate according to any suitable protocol and may include wireless networks, wired networks or fiber optic networks.

[0115] Also, as described, some aspects may be embodied as one or more methods. The acts performed as part of the method may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.

[0116] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

[0117] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” [0118] The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as anon-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0119] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0120] Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having," “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

[0121] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively.

[0122] The terms “substantially”, “approximately”, and “about” may be used to mean within ±20% of a target value in some embodiments, within ±10% of a target value in some embodiments, within ±5% of a target value in some embodiments, within ±2% of a target value in some embodiments. The terms “approximately” and “about” may include the target value. [0123] User of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.